David biweekly meeting
Project 4: Reconstitution of endocytic actin network
recent progress
- finished manually sorting through EM data for images with potential membrane
structures
- see document on Max's laptop; too much data to include here
- started a preliminary quantification of the data
- -Las17: 10 possible vesiculation events out of 46 beads imaged
- +Las17: 29 possible vesiculation events out of 25 beads imaged
- this was split over several sections though; 77 total bead slices
- reached out to Danielle and Emily to meet and get better advice on what is
actually membrane or not
- also asked for references for methods
- checked if we could correlate membrane budding between fluorescence and EM
budding, but that doesn't seem feasible
vesicle I identified by EM
the same vesicle in context of other beads
the same cluster of beads by fluorescence; that vesicle is not visible
next steps
- compare data interpretations with Emily when she finishes hers
- quantify membrane bending/vesiculation with help from Danielle
- write up methods for CLEM
- refresh the manuscript
Project 7: Mechanical regulation of CME by protein LLPS
recent progress
- bugs in my coated membrane model are mostly sorted out now; simulations are
well-behaved
- purple: coat
- black: bare membrane
- red: coat-membrane interface
next steps
- try to replicate conditions of Julian's model to validate that it behaves the same
- build final version of model with protein condensate
Nikhil's protein purification project
project overview
- express and purify His-tagged GFP in E. coli
- insert GFP into His6-eps15 plasmid from Stachowiak lab
- express and purify His6-eps15-GFP from E. coli
- purify His6-eps15
- purify Fcho1
- reproduce phase separation experiments
updates
- while purification was successful, we weren't really observing any induction of our GFP and RFP training constructs
- after lots of troubleshooting the culturing conditions, we realized the constructs we were working with were actually constitutive
- decided it's not worthwhile to continue troubleshooting with the constitutive
constructs, so now working on cloning GFP and GFP-EPS15 into the same vector
that was used to express EPS15 in the Stachowiak lab paper
- we engineered a cleavage site between GFP and EPS15 as a potential alternative purification strategy
Idea for theory rotation project
constant area vs. constant curvature models (Kaksonen and Roux 2018)
- all my CME modeling so far is based on the assumption that the coat is fully polymerized before membrane bending starts (constant area model)
- since the design and analysis of this simple model is already set up, I think
a nice rotation project would be to modify the assumption to constant
curvature instead of constant area and see if that changes the main result
(initial energy barrier)
- the geometry will be slightly different in that case, so it will be important to check
- intuitively though, this shouldn't qualitatively change the main result, but it will probably quantitatively change it